Nanoparticle pharmaceutical delivery system

ABSTRACT

A method of preparing a nanoparticle pharmaceutical delivery system. A nanoparticle pharmaceutical delivery system. A method of preparing a targeted nanoparticle pharmaceutical delivery system. A targeted nanoparticle pharmaceutical delivery system.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Non Provisional patentapplication Ser. No. 16/575,718, which was filed in the U.S. Patent andTrademark Office on Sep. 19, 2019, the entire contents of which areincorporated herein by reference for all purposes.

FIELD OF THE INVENTION

A nanoparticle pharmaceutical delivery system is disclosed.

BACKGROUND

Many small molecule therapeutics, such as taxanes like paclitaxel anddocetaxil, and the platinum derivatives like cisplatin and carboplatin,are highly effective medical therapeutics. However, many small moleculesare difficult to formulate for parenteral administration because oftheir low aqueous solubilities and/or high toxicities. For example, thefirst commercially available formulation of paclitaxel used Cremophor ELas a cosolvent, which has been shown to cause serious side effects suchas hypersensitivity, neurotoxicity, and neuropathy. Subsequently, aprotein-bound PTX formulation (Abraxane) was developed and approved bythe FDA in 2005 and is now widely adopted, though the improvement inefficacy over PTX in Cremophor EL is modest. There exist large librariesof small chemical entities that may, or are known to, have importanttherapeutic benefits, but suffer from the inability to be effectivelyand safely formulated. There is a compelling need for new or alternativeformulations of small molecule therapeutics and potential therapeuticsthat provide stable formulations, reduced toxicity, and improvedefficacy, optionally by targeting the therapeutic to specific cells andtissues within the body that are involved with disease pathophysiology.

SUMMARY

Disclosed is a nanoparticle formulation for delivery of small moleculetherapeutics in a stable, and optionally targeted, delivery platformthat is able to deliver one or more active pharmaceutical ingredients(API) contained in lipid-based nanoparticles that range in size from 30to 225 nanometers in diameter.

More particularly, Applicants' disclosure provides for a stable,pharmaceutically acceptable, nanoparticle solution comprising: 12-28%(v/v) type 1 safflower oil; 1.5-12% (v/v) triacetin; 1.5-5.0% (w/v)surfactant commixture of dipalmitoylphosphatidylcholine anddipalmitoylphosphatidyiglycerol; 0.1 to 0.6% (w/v) therapeuticallyactive small molecule therapeutic, optionally a taxane or a platinumderivate; and 1-4% (w/v) glycerin in buffered water at pH below 8.0.

The solutions comprising Applicants' disclosure are preferably bufferedat a pH of between 4.0 and 7.5. Preferred buffers include tartrate,phosphate, Tris, citrate, or calcium ethylenediaminetetraacetic acid(EDTA). The solutions of this invention preferably contain nanoparticleswhere 95% of the nanoparticles are between 30 and 200 nm in diameter. Itis also preferred that the solutions of this invention contain at leastone small molecule chosen for therapeutic benefit, in concentrationbetween 0.1 and 10.0 mg/ml.

The surfactant commixture or the fully formulated nanoparticle mayoptionally contain a targeting agent directed to a structure on a targetcell. Targeting agents include monoclonal antibodies (mAb), antibodyfragments and peptidomimetics, the latter consisting of molecularentities that possess affinity to specific biological targets of medicalinterest, but are not necessarily proteins. Therapeutic antibodies mayalso be incorporated onto the nanoparticles, either monovalent,bispecific or polyvalent. The invention is exemplified with atherapeutic monoclonal antibody that targets the HER2 receptor on tumorcells, which is a well-known target for cancer therapeutics.

Applicants' disclosure further provides for a method for encapsulating atherapeutically effective amount of a small molecule therapeutic toreduce toxicity and/or improve efficacy in a patient wherein the methodcomprises: encapsulating the API in a nanoparticle where thenanoparticles are in an aqueous solution, the nanoparticle solutioncomprising: 12-28% (v/v) type 1. safflower oil; 1.5-12% (v/v) triacetin;1.5-5.0% (w/v) surfactant commixture of dipalmitoylphosphatidylcholineand dipalmitoylphosphatidylglycerol; 0.1 to 0.6% (w/v) therapeuticallyactive API, 1-4% (w/v) glycerin in buffered water at pH below 8.0. Thepreferred APIs, buffers, nanoparticle sizes and concentration of taxanesare as described above for the solutions. The components of thesurfactant commixture and targeting agents are also as provided abovefor the solutions of this invention.

Applicants' disclosure also provides for a method of delivering atherapeutically effective amount of an API to a patient in need thereof,where the method comprises the step of administering the therapeuticallyeffective amount of API in a stable, pharmaceutically acceptable andwell-tolerated nanoparticle solution, the solution comprising: 12-28%(v/v) type 1 safflower oil, 1.5-12% (v/v) triacetin, 1.5-5.0% (w/v)surfactant commixture of dipalmitoylphosphatidylcholine anddipalmitoylphosphatidylglycerol; 0.1 to 0.6% (w/v) therapeuticallyactive API, 1-4% (w/v) glycerin in buffered water at pH below 8.0. Thepreferred APIs, buffers, nanoparticle sizes and concentration of API areas described above for the solutions. The components of the surfactantcommixture and targeting agents are also as provided above for thesolutions of Applicants' invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically recites a histogram showing particle number as afunction of size;

FIG. 2 graphically recites body weight changes, and tumor volumechanges, over time for each treatment group identified;

FIG. 3 graphically recites body weight changes, and tumor volumechanges, over time for each treatment group identified;

FIG. 4 graphically recites body weight changes, and tumor volumechanges, over time for each treatment group identified. The cisplatinnanoparticle formulation (labeled HJ9 in FIG. 4) effected a significantreduction in tumor growth, as did the positive control of freecisplatin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Applicants' nanoparticle pharmaceutical delivery system is described inpreferred embodiments in the following description with reference to theFigures, in which like numbers represent the same or similar elements.Reference throughout this specification to “one embodiment,” “anembodiment,” or similar language means that a particular feature,structure, or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” andsimilar language throughout this specification may, but do notnecessarily, all refer to the same embodiment.

The described features, structures, or characteristics of Applicants'disclosure may be combined in any suitable manner in one or moreembodiments. In the following description, numerous specific details arerecited to provide a thorough understanding of embodiments of theinvention. One skilled in the relevant art will recognize, however, thatApplicants' disclosure may be practiced without one or more of thespecific details, or with other methods, components, materials, and soforth. In other instances, well-known structures, materials, oroperations are not shown or described in detail to avoid obscuringaspects of Applicants' disclosure, and it will be appreciated by thoseskilled in the art that it is intended to cover alternatives,modifications, and equivalents as may be included within the spirit andscope of Applicants' disclosure as defined by the appended claims andtheir equivalents as supported by the following disclosure and drawings.

In certain embodiments, Applicants disclose a method to form aNanoparticle Pharmaceutical Delivery System and a NanoparticlePharmaceutical Delivery System formed by Applicants' method. In certainof these embodiments, both the method, and the composition formed by themethod, utilize/comprise one Active Pharmaceutical Ingredient (“API”).In other of these embodiments, both the method, and the compositionformed by the method, utilize/comprise more then one ActivePharmaceutical Ingredient (“API”).

In certain embodiments, Applicants disclose a method to form a TargetedNanoparticle Pharmaceutical Delivery System and a Targeted NanoparticlePharmaceutical Delivery System formed by Applicants' method. In certainof these targeted embodiments, both the method, and the compositionformed by the method, utilize /comprise one Active PharmaceuticalIngredient (“API”). In other of these targeted embodiments, both themethod, and the composition formed by the method, utilize /comprise morethen one Active Pharmaceutical Ingredient (“API”).

A. Introduction

Many small molecules, such as taxanes and platinum derivatives, arewidely used chemotherapeutic agents for the treatment of cancer, owingto their potency as anti-tumor agents for many different cancer types.However, despite their widespread use they suffer from severallimitations that reduce their efficacy and safety. First, many smallmolecules, such as the taxanes, and especially paclitaxel, have verypoor solubility in aqueous media such as buffers and blood plasma.Current methods to solubilize such APIs include cosolvents, which may betoxic and cause hypersensitivity reactions in many patients.

More broadly, there exist many small molecules that are demonstrated tohave desired activities on models of human diseases, but suffer from aready means to be formulated for safe administration in patients. Insome cases, lack of aqueous solubility prevents the small molecule frombeing safely delivered to patients, sometimes requiring toxic excipientsto aid solubility. In other cases, potential small molecule therapeuticscannot be delivered to the appropriate sites of action within a patient,and thus are rendered less effective. Biodistribution of therapeuticformulations is a frequent challenge in the therapeutic setting,especially for lipophilic small molecules that partition into lipid-richvolumes of the human body and are thus unavailable therapeutically. Forthese reasons it is desirable to have a formulation system that iscapable of containing therapeutic small molecules such that they aredistributed to areas or volumes within the body involved in the disease.

Many currently approved formulations are not targeted, relying insteadon passive biodistribution to reach the site of intended activity inadequate amounts to be clinically effective. Newer attempts atnanoparticle formulations, some of which are targeted, suffer from aninability to load clinically significant or optimal amounts of API ontothe nanoparticles, rendering them inadequate as therapeutics. Currentlyavailable targeted therapeutics do not include a therapeutic antibody,either as a single therapeutic entity or in combination with otherpharmacologically active molecules.

From a pharmacologic perspective, both nanoparticle size and stabilityare important. Since therapeutics are formulated remotely and stored forindefinite periods of time before administration, it is important thatany nanoparticle formulation remain stable for the duration of timebetween formulation and use to insure that the therapeutic is given asspecified. Breakdown of nanoparticles resulting in non-homogeneousmixtures not only lose efficacy, but can actually be dangerous. Forexample, large pools of lipids can cause transient hypoxia in vitaltissues such as those found in the nervous system and in cardiac muscle.This can cause serious side effects such as embolic stroke andmyocardial infarction.

The size of the nanoparticles is important for at least two reasons.First, parenterally administered nanoparticle formulations are initiallyintroduced into the body through the vasculature. However, many diseasetargets occupy volumes outside any blood vessels. Thus it is importantfor intravascular therapeutics to be small enough to extravasate throughjunctions in the vasculature to reach the site of intended activity.Although this process is helped, for example, by the inherent leakinessof the angiogenic vessels that surround tumors, larger particles (forexample, greater than 200 nanometers in average diameter) havecommensurately less tendency to pass from the plasma to the interstitialspace around the tumor, and thus commensurately lower efficacy. Inaddition, larger average diameter nanoparticles are generally lessstable and may decompose in plasma, which may cause similar problems toinhomogeneous lipid mixtures described above.

In the disclosure herein, methods and compositions of stable andappropriately sized API-containing nanoparticle formulations arepresented that allow distribution of therapeutically efficacious amountsof API. Applicants' formulation comprises one or more lipids, at leastone of which is chosen for its ability to dissolve the API and anotherof which is chosen for its ability to form stable lipid nanoparticleswhen combined with certain surfactants. In many cases, both lipids arethe same. The addition of an optional lipid-anchored targeting ligandwith high affinity to surface proteins selectively expressed on manycancer cells and the surrounding tumor vasculature, or other diseasetargets, provides a means to target the API-containing nanoparticles tocells and tissues directly involved with many cancers, as well asprovide therapeutic benefit, for example, though the use of atherapeutic mAb as the targeting ligand.

B. Definitions

Lipid: As used herein, a lipid refers to a non-polar hydrophobicmolecule or a plurality of molecules characterized by longer chainhydrocarbons. Lipids may be comprised of mixtures of one or more fattyacids like oleic, linoleic, stearic and/or palmitic acids. For example,safflower oil is a naturally produced mixture of fatty acids and smallamounts of other components, which is commonly used for parenteralnutrition and other medical uses. Another example is triacetin, thetri-ester of glycerol and acetic acid, which dissolves small lipophilicmolecules such as the taxanes, and is also routinely used for parenteralnutrition.

Surfactant: As used herein, a surfactant is an amphipathic molecule thatincludes a polar, hydrophilic domain, and a non-polar, hydrophobicdomain. Examples include the large class of amphipathic compounds knownas phosopholipids, which have a phosphate group that comprises the polardomain, and one or more long chain fatty acids that comprise thenon-polar domain.

Nanoparticle: As used herein, a nanoparticle refers to asurfactant-stabilized lipid-based particle with a mean and medianparticle diameter between 60 nanometers and 225 nanometers when residingin an aqueous buffer or media.

Emulsion: As used herein, an emulsion refers to a combination of lipidsand surfactants in an aqueous buffer or media. Specific subtypes ofemulsions include crude emulsions, in which the aqueous and lipid phasesare inhomogeneously and/or incompletely mixed; and fine emulsions, inwhich most or all of the lipid components are contained insurfactant-stabilized nanoparticles that are homogeneously distributedin an aqueous buffer.

Active Pharmaceutical Ingredient (API): As used herein, an API is one ormore molecular entities that have desired pharmacologic and therapeuticactivities. An API may be comprised of a small molecule of synthetic ornatural origin, a protein or fragment thereof, therapeutic antibodiesand fragments thereof, or other biologic or chemical entity that isknown to, or suspected of, having pharmacologic activity. Examples ofAPIs include paclitaxel, docetaxel, other taxane derivatives, andcisplatin and other platinum derivatives.

Targeting ligand: As used herein, a targeting ligand is a molecularstructure that is attached to the outermost volume of the nanoparticle,optionally in the surfactant layer that stabilizes the nanoparticles,and has binding affinity to a specific class of in vivo biologicstructures or targets of therapeutic interest. The targeting ligand maybe a small molecule, a peptidomimetic, an antibody or antibody fragment,optionally chosen for pharmacologic activity, a protein, or othermolecular entity.

Target: As used herein, a target refers to a naturally occurring in vivostructure that possesses affinity to the targeting ligand. A target mayinclude proteins that are expressed on the surfaces of certainbiological cells, such as cancer cells and endothelial cells in thevasculature surrounding a tumor. An example of a target is the HER2receptor, a protein that is selectively expressed on the surface ofcertain cancer cells.

C. Stable Pharmaceutically Acceptable Nanoparticle Solutions

The materials used in the invention are comprised of lipids,surfactants, buffers and active pharmaceutical ingredients (APIs), asdefined above. In general, the choice of lipids depends on the API beingused, as different APIs may require different lipids to be dissolved. Inthe case of the taxanes, triacetin is an example of a lipid that workswell to dissolve the API. The other factor affecting choice of lipid isnanoparticle stability. As described in detail in example 6, many lipidsdo not form stable nanoparticles; and of those that do, many do notdissolve the API. In addition, certain lipids that form stablenanoparticles alone will not do so if they are mixed with a solubilizinglipid such as triacetin. Thus, in accordance with the inventiondescribed herein, the following materials are presented in order to morecarefully describe the methods and composition of this class ofnanoparticle formulations.

Materials

Type 1 safflower oil: Type 1 safflower oil is a naturally producedplant-based lipid that contains a predominance of linoleic acid,typically in the range of 70-85% of the total lipid content of saffloweroil. Other major lipids include oleic acid, stearic acid, and palmiticacid. Other minor constituents may also be present in safflower oil.USP-grade safflower oil may be purchased by vendors such as Croda, Inc.

Triacetin: This nine-carbon lipid is the tri-ester of glycerol andacetic acid, and is the principal lipid responsible for dissolving thetaxanes. USP-grade triacetin can be purchased from a number of vendors,including Sigma-Aldrich, Inc., and Spectrum Chemical, Inc.

Surfactant commixture: The surfactant commixture is comprised of amixture of surfactants. The individual components of the commixture mayinclude lecithin (Lipoid GmbH, Ludwigshaven, DE), and1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG, Lipoid GmbH,Ludwigshaven, DE). The surfactant commixture may optionally contain atargeting ligand, such as the a_(v)r33-targeting peptidomimeticdescribed below, or it may contain a surfactant that binds anappropriately prepared targeting ligand, such as DSPE-PEG-biotin, whichbinds a mAb that has streptavidin conjugated to the Fc portion of theantibody.

Taxane derivatives: Taxanes may optionally be used as the API. Taxanesare diterpines that are produced by plants of the genus Taxus. They area class of small molecules that are widely used in cancer chemotherapyand other medical uses. Taxanes include, but are not limited to,compounds that are structurally similar to or are in the same generalchemical class such as paclitaxel (i.e., taxol), docetaxel (i.e.,taxotere), or ortataxel, and pharmaceutically acceptable salts,derivatives, or analogs of paclitaxel, docetaxel, and ortataxel.

Their mechanism of action is to stabilize microtubules in dividingcells, effectively preventing cell division by blocking disassembly ofthe microtubules. Paclitaxel was the first taxane to be purified andused as a chemotherapeutic agent, and is still widely used. Docetaxel isa more recent taxane, which has also found widespread use as a cancerchemotherapeutic agent. In 2010 Cabazitaxel, another taxane derivative,was approved for use in certain cancers. Purified USP-grade paclitaxeland docetaxel may be purchased from a number of vendors, includingTecoland, Inc.

Buffers: The aqueous phase of the formulation is comprised of a buffersuch as tartrate. These buffers may be purchased pre-mixed(Sigma-Aldrich) or formulated from purified water (Calbiochem) and theindividual constituents, including tartaric acid (J. T. Baker), sodiumphosphate monobasic monohydrate (Mallinckrodt), glycerin (Mallinckrodt),hydrochloric acid (VWR), and sodium hydroxide (VWR).

Targeting ligand: As defined previously, a targeting ligand is amolecular structure that has binding affinity to a specific class of invivo biologic structures of therapeutic interest. One such targetingligand used in the invention described herein is a monoclonal antibody(mAb) that is chosen for its specificity to a desired target. Forexample, a mAb specific for HER2, where specificity is defined as havingat least four-fold greater affinity for HER2 than for related orunrelated protein targets, is used to target the nanoparticle to theHER2 receptor that is highly expressed on certain cancer cells. Otherexamples include peptidomimetics, fragments of monoclonal antibodies,and other proteins and small molecules that have specific affinities forbiological targets. The targeting ligand may be incorporated into thenanoparticle during the formulation of the fine emulsion, for example byincluding a peptidomimetic conjugated to a surfactant, or the targetingligand may be incorporated into the nanoparticle post-formulation, forexample by incorporating at least one streptavidin molecule onto the Fcportion of the mAb and including a biotinylated surfactant in theformulation.

Process for making

Formulation begins by dissolving the API, for example a taxane, in alipid that dissolves the API, for example triacetin in the case of ataxane, using heat and sonication. A crude mixture of the API in thesolvent or suspending lipid at concentrations below the molar solubilityof the API in the lipid is heated by a water-bath and the API isdissolved or suspended using sonication. This process continues untilthe API precipitate is entirely dissolved in the solvent lipid, asevidenced by the lack of any precipitate in the mixing vessel and aclear appearance of lipid.

Next, the individual components of the surfactant commixture aresuspended in an appropriate buffer containing glycerin with high-sheardispersion, using any of the readily available high-shear laboratorymixers (for example an Ultra-Turrax high shear mixer available fromHomogenizers, Inc.). This is followed by addition of the safflower oiland the API/lipid solution, followed by additional high-shear mixing.

This crude emulsion is then processed into a fine emulsion using ahigh-pressure microfluidizer such as a M110Y or M110EH Microfluidicsemulsifier (Microfluidics, Newton, Mass.) at approximately 10,000-25,000pounds per square inch for a predefined number of passes through thefluidic circuit, until a fine emulsion of nanoparticles has beenachieved. The number of passes is determined by first measuring the flowrate. The volume of the crude emulsion is then multiplied by the desirednumber of passes and then finally divided by the previously determinedflow rate to yield a time duration of processing that will result in thedesired number of passes. If a fine emulsion is not achieved with aninitial phase of processing, the number of passes may be extended toachieve the desired formulation, optionally by monitoring the particlesize in real time and adjusting and/or curtailing the process after thedesired particle size is achieved.

During the microfluidization (emulsification) process the emulsion iskept cool by routing part of the fluidic circuit through a cooling bath.For example, a VWR Model 1175MD refrigerated circulator filled with 1:1glycol/water can be connected to the Process Fluid Heat Exchanger of theM-110EH Microfluidizer to maintain the temperature of the emulsion near5° C. The combination of high pressure and high shear fluid flow tendsto heat up the emulsion, which can limit the formation of nanoparticlesand/or degrade existing nanoparticles.

Once the emulsion is formed it is initially characterized according toparticle size using dynamic or static light scattering methods on acalibrated analytic system intended for such uses (for example a HoribaLA-910 static light scattering particle size distribution analyzer).Typical desired particle sizes span the mean and median range from 60nanometers to 225 nanometers, without appreciable evidence of anyparticle populations greater than 1 micrometer. Additionalcharacterization includes visual inspection for homogeneity, formationof precipitates and pooling of lipids. The concentration of the API inthe processed fine emulsion is measured using high-performance liquidchromatography (HPLC) or inductively coupled plasma mass spectrometry(ICP-MS). A standard curve is run on the HPLC, followed by test samplesof the fine emulsion to determine the amount of API present.

Once the fine emulsion is formulated, a targeting ligand is optionallyattached to the outermost portion of the nanoparticles using theappropriate binding chemistries. Such chemistries include the ‘click’chemistries, as is well known in the art, or it may utilize the verystrong biotin-streptavidin reaction commonly used in many areas ofbiology and chemistry. The latter example has the benefit of simplicityand the lack of additional reagents or reaction conditions to achievebinding.

Once the processed emulsion passes the initial characterizationcriteria, as outlined previously, it is placed into appropriately sizedcontainers and sterilized. The sterilization method is optionally chosento be either heat and pressure sterilization using available equipmentdesigned for this purpose; or using the process of sterile filtrationusing 32 mm Pall Acrodisc PF syringe filters with 0.2 [tm Supormembranes, in which the emulsion is passed through a size-based filterthat is chosen such that the nanoparticles pass freely butmicroorganisms do not. The latter method may be preferable in caseswhere a protein or antibody fragment is used for the targeting ligand.Optionally, the formulation is sterilized before conjugating thetargeting ligand.

Additional follow-up characterization of particle size and amount of APIpresent is done after sterilization to insure nanoparticle integrity,emulsion stability, and to quantitate the amount of API. Relativelysmall increases in mean and median diameters in the first week afterformulation are acceptable (<20%), but continued increases in diameterbeyond the first week, or the appearance of particles greater than 1micrometer in diameter at any time, indicate instability in theformulation.

Presence and quantity of the API are measured using any of thewell-known methods for bioanalysis, such as mass spectrometry or HPLC.In the case of HPLC, reference standards for calibration are preparedand used to quantitate the results of the HPLC analysis on theformulation. HPLC analysis measures the amount of API as a percentage ofan ideal recovery. For example, if 2.0 mg/ml of paclitaxel istheoretically incorporated into a nanoparticle formulation, anacceptable recovery might be 1.8 mg/ml, for a 90% recovery. Recoveriesof the API are used to determine dosing levels for subsequent studies inwhich the analyzed formulation is used. In addition, low recoveries(<60%) may indicate a formulation problem. It is also useful toperiodically analyze archival amounts as representative of a batch offormulation that is used in in vivo or in vitro studies, to determinethe actual amounts of API being used in the studies and to determine ifthe API is coming off the nanoparticles.

If the nanoparticles are optionally targeted, it is possible to verifythat the nanoparticles have the desired affinity to their intendedtarget using the appropriate method. For example, a fluorescent dye suchas rhodamine may be incorporated into the nanoparticle, and theresulting nanoparticles are then exposed to cells that express theintended target, for example in a flow cytometer. A comparison may thenbe made between targeted and untargeted nanoparticles, and between cellsthat express the target and cells that do not. A pre-defined differencein fluorescence, for example a three-fold difference, is used todetermine suitable affinity for the nanoparticles to the intendedtarget.

Finally, formulations are also characterized by visual inspection toinsure homogeneity, lack of precipitates and pooling or separation oflipids from the aqueous buffer.

Once the individual containers are sealed and sterilized, they arestored at approximately +5° C., but not frozen.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those skilled in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Formulation of a HER2-Targeted Paclitaxel-ContainingNanoparticle Emulsion

In the following example we provide the steps for preparing aHER2-targeted nanoparticle emulsion of paclitaxel.

The reagents used for this exemplary formulation, along with the sourcesfor these materials, are listed in table 1 below.

TABLE 1 Reagent Manufacturer Water for Injection (WFI) CalbiochemTartaric Acid, Granular J.T. Baker Sodium Phosphate MonobasicMonohydrate Mallinckrodt Glycerin Mallinckrodt 1N Hydrochloric Acid(HCl) VWR 1N Sodium Hydroxide (NaOH) VWR EPCS Egg Lecithin LipoidDipalmitoylphosphatidylglycerol (DPPG) NOF mPEG-DSPE, MW 1000 Nano-softPolymers DSPE-PEG-biotin, MW = 2000 Nanocs Targeting Ligand (HER2 mAbconjugated to BioLegend streptavidin) Safflower oil Spectrum TriacetinSpectrum Paclitaxel Tecoland

To prepare the nanoparticle solution in this example, the followingequipment was used:

Ultra-Turrax high-shear mixer.

Microfluidics M-110Y Microfluidizer to process crude emulsion to givefine emulsion.

Sterile filtration using a Pall Acrodisc PF filters w/ 0.2 [tm Supormembranes.

Horiba LA-910 static light scattering particle size distributionanalyzer for particle size determination.

A tartrate buffer was first prepared by dissolving 1.13 grams oftartaric acid in 1 liter water for injection (WFI) with stirring viamagnetic stirrer. Next, 22 grams of glycerin were transferred to thesolution using a pipette, then stirred. The pH of the buffer was broughtto 7.0 with 1 N NaOH, as measured using Orion 720 A+pH meter equippedwith a ROSS Ultra pH electrode, and placed in a 1 liter volumetricflask. The final volume was brought to 1 liter using WFI.

Next, the 250 mg of paclitaxel was solubilized in 5 ml of triacetin bywater-bath sonication at 50° C., using 50 milligrams of paclitaxel perone milliliter of triacetin. The paclitaxel was heated and sonicateduntil there was no more visible precipitate and the solution wastransparent.

Next, the surfactant commixture was prepared. The components of thesurfactant commixture included 1.40 g Lipoid EPCS eggphosphatidylcholine (Lipoid GmbH, Ludwigshaven, Del.), 0.60 g of1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG, NOF Corporation,Tokyo, JP), 150 mg of DSPE-PEG-biotin 1\4W 2000 (DSPE-PEG2K-biotin,Nanocs Inc., New York, N.Y.) and 5 mg of mPEG-DSPE MW 1000 (DSPE-PEG1K,Nanosoft Polymers, Winston-Salem, N.C.). These components were pre-mixedin 32 mL of pH 7.0 tartrate buffer with 22 mg/mL glycerin in a 100 mLdisposable beaker using the Ultra-Turrax high shear mixer for 30seconds. This was followed by addition of 13 ml of Type 1 safflower oiland the paclitaxel/triacetin solution prepared above, followed by anadditional 2 minutes of mixing on the high shear mixer to give the crudeemulsion.

This crude emulsion was then further emulsified on a M110Y Microfluidicsemulsifier (Microfluidics, Newton, Mass.) at approximately 15,000 poundsper square inch for 50 passes, using the method described previously.The temperature of processing was maintained at approximately 5° C. byusing an ice bath to cool the Process Fluid Heat Exchanger of theMicrofluidizer.

The emulsion was buffered to a pH of 7.0, and characterized by particlesize and distribution via static light scattering (FIG. 1). The meanparticle size was found to be 80 nm, without evidence of particleslarger than 200 nm. There were no visible signs of lipid pooling orphase separation, and the fine emulsion appeared homogeneous. Theprocess above resulted in approximately 50 ml of fine nanoparticleemulsion.

The 50 ml volume of the fine emulsion was sterile filtered using PallAcrodisc PF filters w/ 0.2 [tm Supor membranes, followed by a particlesize measurement and HPLC analysis. It was then placed into multipleWheaton serum sample vials, each with 5 mL of emulsion and—4 mLheadspace, followed by nitrogen sweep, capping and sealing (gray butylrubber caps with Flip-Off seals, obtained from West Pharmaceuticals).The mean particle size had not changed after sterile filtration, andHPLC analysis confirmed a recovery of 89% of the paclitaxel.

Samples were stored at 2-8° C., not frozen, and characterized weeklythereafter via static light scattering and HPLC. Mean particle diameterdid not increase with time, and subsequent HPLC analyses showedconsistent recoveries in the range of 90%.

A targeting ligand in the form of an anti-HER2 mAb, a therapeuticantibody, was conjugated to the above nanoparticle formulation asfollows: A vial containing a 4 mg/ml concentration of anti-HER2 mAb withat least one streptaviden (SAV) molecule covalently connected to the Fcportion of the majority of antibodies was purchased from BioLegend, Inc.This vial, along with the biotinylated emulsion and all the necessaryequipment, as described below, was wiped into a Class 5 hood at roomtemperature using 70% EtOH. Using a cross-shaped magnetic stirring barthat had been wiped into the hood, the biotinylated emulsion was stirredat medium speed on a magnetic stir plate that had been wiped into thehood. Using a transfer pipette that has been wiped into the hood, anamount of the SAV-mAb conjugate was transferred to a sterile 10 mLburette with a PTFE stopcock, the amount chosen was such that the finalconcentration when added to the emulsion would be 0.295 mg/ml. Thefilled burette was then clamped in place above the vial containing thebiotinylated emulsion that was being stirred. The SAV-mAb conjugate inthe burette was then added drop-wise into the biotinylated emulsion,until the burette was empty. The emulsion was then stirred for anadditional ten minutes. Stirring was then discontinued, and the magneticbar was removed using a magnet that had been wiped into the hood. Thevial was swept with purified nitrogen and sealed with pre-sterilizedstopper (West Pharma). The vial was then removed from the Class 5 hoodand sealed with an appropriately sized Flip-Off seal. The vial was thenplaced on a carousel inverter at room temperature and continuouslyinverted at low speed for two hours. The vial was then stored between2-8° C.

Example 2 Characterization of Nanoparticle Formulation

A paclitaxel nanoparticle emulsion was prepared substantially asdescribed in example 1, with 5.0 milligrams of paclitaxel per milliliterof fine nanoparticle emulsion, then terminally sterilized using heat andpressure and stored at +5° C. for one week. The distribution of particlesizes, including mean and median particle sizes, were determined using astatic light scattering particle size analyzer (Horiba Instruments,Inc., Irvine, Calif.). The particle sizes showed a mean and mediandiameter of approximately 80 nanometers (FIG. 1). The distribution ofparticle sizes was narrowly and symmetrically distributed around themean and median diameter, with no populations of particles larger than250 nm, indicating a stable fine emulsion of nanoparticles.

Presence and quantity of the API was measured using high-performanceliquid chromatography (HPLC), as described previously. The recoveryafter one week was 86.2%, indicating adequate API on the nanoparticlesand good stability.

Finally, the nanoparticle formulation was characterized by visualinspection, and noted to be homogeneous, without precipitates or visiblephase separation.

Example 3 In Vivo Efficacy and Toxicity of an Exemplary TargetedPaclitaxel-Containing Nanoparticle Emulsion

To demonstrate the therapeutic potential of the invention describedherein, the following studies were carried out in mouse xenograft modelsof cancer.

Animal Models: Female athymic nude mice (Crl:NU(Ncr)-Foxnlnu, CharlesRiver) that were eight weeks old and had a body weight (BW) range of19.0 - 24.8 grams (g) on Day 1 of the study were used. The animals werefed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified andIrradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat,and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'cobs™Laboratory Animal Bedding in static microisolators on a 12-hour lightcycle at 20-22 ° C. (68-72 ° F.) and 40-60% humidity.

Tumor Cell Culture: MDA-MB-231 breast cancer cells were grown to mid-logphase in RPMI 1640 medium containing 10% fetal bovine serum, 2 mMglutamine, 100 units/mL sodium penicillin G, 25 μg/mL gentamicin, and1001.tg/mL streptomycin sulfate. The tumor cells were cultured in tissueculture flasks in a humidified incubator at 37 ° C., in an atmosphere of5% CO2 and 95% air.

Tumor Implantation and Measurement: The MDA-MB-231 cells were harvestedduring exponential growth and resuspended cold in PBS. Each test animalreceived a subcutaneous (s.c.) injection of 5×10⁶ MDA-MB- 231 cells (0.1mL cell suspension) into the right flank, and tumor growth was monitoredas the average tumor size approached the target range of 100 - 150 mm³.Tumors were measured in two dimensions using caliper measurement oflength and width.

Eighteen days after tumor cell implantation, on Day 1 of the study, micebearing appropriately sized tumors were sorted into six groups of tenmice each (n=10). The individual tumor volumes range from 75 to 144 mm³and the group mean tumor volumes were 106-108 mm³.

Referring now to FIG. 2, two _(a)∘33-targeted paclitaxel-containingnanoparticle emulsions were prepared and fully characterized. Targetingwas achieved by inclusion of a peptidomimetic that is specific for thea_(v)33-integrin, a cell-cell adhesion protein that is highly expressedin the peritumoral neovasculature. The amounts of paclitaxel containedin each emulsion were determined using the HPLC methods describedpreviously. A first emulsion (KLN-0092-030) contained paclitaxel at 2.69mg/ml, and a second emulsion (KLN-0092-029) contained paclitaxel at 1.82mg/ml.

For a positive control, paclitaxel was obtained in a dry powder formfrom Phyton Biotech, LLC (Fort Worth, Tex.). A paclitaxel stock solution(30 mg/mL) in 50% ethanol: 50% Cremophor EL was prepared and storedprotected from light at room temperature during the dosing period. Oneach day of dosing, an aliquot of the paclitaxel stock was diluted with5% dextrose in water (D5W) to yield a 3.0 mg/mL paclitaxel dosingsolution in a vehicle consisting of 5% ethanol: 5% Cremophor EL: 90% D5Win a 10 mL/kg dose volume.

Study Design: Six groups of ten NCr nu/nu mice were dosed according tothe protocol outlined below. Doses were adjusted accordingly for bodyweights and were given by slow i.v. push. When doses were above 10 mL/kgthey were split into two parts dosed 30 minutes apart.

Group 1 received no treatment and served as the benchmark group fortumor engraftment and progression as well as a negative control. See,Curve 210 FIG. 2).

Group 2 received KLN-0092-029 administered intravenously (i.v.), at 20mg/kg, on Days 1 and 3, then 30 mg/kg on Days 5, 7 and 9, followed by 40mg/kg on Day 15. These doses were given at 10.99, 16.48 and 21.98 mL/kgrespectively. See, Curve 220, FIG. 2)

Group 3 received KLN-0092-029 i.v., at 40 mg/kg, on Days 1 and 3,followed by 30 mg/kg on Days 5, 7 and 9. These doses were given at 21.98and 16.48 mL/kg respectively. See, Curve 230, FIG. 2).

Group 4 received KLN-0092-030 i.v., at 20 mg/kg on Days 1 and 3, then 30mg/kg on Days 5, 7 and 9, followed by 40 mg/kg on Day 15. These doseswere given at 7.43, 11.15 and 14.87 mL/kg respectively. See, Curve 240,FIG. 2).

Group 5 received KLN-0092-030 i.v., at 40 mg/kg, on Days 1 and 3,followed by 30 mg/kg on Days 5, 7 and 9. These doses were given at 14.87and 11.15 mL/kg respectively. See, Curve 250, FIG. 2).

Group 6 received paclitaxel, i.v., at 30 mg/kg, every other day for atotal of five doses. See, Curve 260, FIG. 2). p Toxicity: Animals wereweighed daily for the first five days of the study and twice weeklythereafter. The mice were observed frequently for overt signs of anyadverse, treatment-related (TR) side effects, and clinical signs oftoxicity were recorded when observed. Individual body weight (BW) losswas monitored as per protocol and any animal that exceeded the limitsfor acceptable BW loss was euthanized. Group mean body weight loss alsowas monitored as per protocol. Acceptable toxicity was defined as agroup mean BW loss of less than 20% during the study and not more than10% mortality due to TR deaths. Any dosing regimen resulting in greatertoxicity was considered above the maximum tolerated dose (MTD). A deathwas classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy, or if due to unknown causesduring the dosing period or within fourteen days of the last dose. Adeath was classified as non-treatment-related (NTR) if there was noevidence that death was related to treatment side effects.

FIG. 2 shows the percent change in mean body weight for each group,based on the mean body weight for each group on the first day of dosing.No group reached the 20% reduction of body weight threshold fortoxicity. There were no overt signs and symptoms of toxicity, and therewere no treatment-related deaths on non-treatment-related deaths in anyof the groups.

Note that the positive control was paclitaxel in a standard Cremophor ELformulation. The toxicity of Cremophor EL is sporadic and not alwaysevident in animal models. The positive control was dosed within awell-established maximum tolerated dose, which was chosen so thatminimal side effects and toxicities occur. Further, the dosing of thenanoparticle emulsion was very conservative, at about half thepreviously determined maximum tolerated dose in healthy normal mice.This was done to insure that most of the weakened tumored mice survivedthe study without unexpected toxicity or mortality. Because of this,there is no expectation of demonstrating superior toxicity profiles foreither of the two nanoparticle emulsions over the conventionallyformulated paclitaxel.

Efficacy: Using the caliper method of measuring tumor size, as describedabove, the mean tumor size of each treatment group was followed for 60days. The mean tumor size for each of these groups is shown in FIG. 3.In all cases, the a_(v)r33-integrin-targeted paclitaxel-containingnanoparticle arms showed a reduction in tumor size substantially similarto the positive control, and statistically very different (p<0.001) fromthe untreated arm. There were no treatment-related ornon-treatment-related deaths in any of the nanoparticle emulsion arms.

Example 4 Demonstration of Targeting Effect

To demonstrate the benefit of targeting the nanoparticle emulsion, astudy on A549 lung cancer mouse xenograft cancer models was carried outin which both targeted and untargeted nanoparticle emulsions werecompared to positive (conventionally formulated paclitaxel) and negative(untreated) controls.

The study methods, design, and toxicity criteria were similar to what ispresented in example 3, with the following exceptions: an A549 lungcancer xenograft model was used, and targeting was achieved by inclusionof peptidomimetic, TL-29, that is specific for the a_(v)r33-integrin, acell-cell adhesion protein that is highly expressed in the peritumoralneovasculature. TL-29 is not known to be pharmacologically activeagainst any cancers.

Formulation for the targeted emulsion was achieved as follows:

1.13 g tartaric acid (J. T. Baker) was dissolved in approximately 950 mLsterile water for injection (Baxter) in a 1 L beaker using magneticstirring. Next 22.00 g glycerin (Mallinckrodt) was added with stirring.The pH of the buffer solution was then adjusted to final pH of 4.50using 1 N sodium hydroxide (VWR). Finally the solution was brought to afinal volume of 1 L using sterile water for injection (Baxter).

Next, 213.6 mg of paclitaxel (PTX; Tecoland) was dissolved in 6 mL oftriacetin (Sigma) by rotating a 25 mL single neck round bottom flaskcontaining the paclitaxel and triacetin in a heated (41° C.) sonicatingbath for 30 min using a Büchi R-200 Rotavapor.

2.14 g of purified egg phosphatidylcholine (Lipoid) and 10.6 mg ofa_(v)r33-TL 29 targeting ligand, a petidomimetic (Kereos;KLN-0062-095B1) were homogenized in 84 mL of pH 4.50 7.5 mM tartratebuffer with 22 mg/mL glycerin for 30 seconds in a 250 mL disposablebeaker using an Ultra-Turrax T25 rotor stator homogenizer. Next, 17 mLof safflower oil (Sigma) was added to the contents of the beaker,followed by the addition of the PTX/triacetin solution, and the combinedcomponents homogenized for an additional 2 minutes. The resulting crudeemulsion was then processed on a M-110-EH Microfluidizer (Microfluidics)at approximately 20,000 psi for 13 minutes at a temperature of 5 ° C.and flow rate of 410 mL/min. A post-processing particle size measurementshowed a distribution with a median of 0.1525 [tm and a mean of 0.1693[tm with a small population (0.7%) ranging from approximately 0.15 [tmto approximately 0.3 [tm. The fine emulsion was then extruded throughtwenty 0.45 [tm syringe filters (Whatman Puradisc 25 PP syringe filterswith 0.45 [tm polypropylene media) using a 60 mL disposable syringe(BD). The filtered emulsion was then vialed in Wheaton 5 mL serum samplevials with a 5 mL fill and stoppered and sealed with rubber stoppers andFlip-Off seals (West Pharmaceutical). Post formulation HPLC analysisshowed the paclitaxel concentration at 1.85 mg/mL.

A non-targeted formulation was made as follows:

1.13 g tartaric acid (J. T. Baker) was dissolved in approximately 950 mLsterile water for injection (Baxter) in a 1 L beaker using magneticstirring. Next 22.00 g glycerin (Mallinckrodt) was added with stirring.The pH of the buffer solution was then adjusted to final pH of 4.50using 1 N sodium hydroxide (VWR). Finally the solution was brought to afinal volume of 1 L using sterile water for injection (Baxter).

240.0 mg of paclitaxel (PTX; Tecoland) was dissolved in 7 mL oftriacetin (Sigma) by rotating a 25 mL single neck round bottom flaskcontaining the paclitaxel and triacetin in a heated (41° C.) sonicatingbath for 1 hour and 15 min using a Büchi R-200 Rotavapor.

240g of purified egg phosphatidylcholine (Lipoid) was homogenized in 94mL of pH 4.50 7.5 mM tartrate buffer with 22 mg/mL glycerin for 30seconds in a 250 mL disposable beaker using an Ultra-Turrax T25 rotorstator homogenizer. Next 19 mL of safflower oil (Sigma) was added to thecontents of the beaker, followed by the addition of the PTX/triacetinsolution and the combined components homogenized for an additional 2minutes. The resulting crude emulsion was then processed on a M-110-EHMicrofluidizer (Microfluidics) at approximately 20,000 psi for 14minutes 30 seconds at a temperature of 5 ° C. and a flow rate of 410mL/min. A post-processing particle size measurement showed adistribution with a median of 0.1462 [tm and a mean of 0.1634 [tm with asmall population (0.7%) ranging from approximately 1 μm to approximately2 μm. The fine emulsion was then extruded through twenty 0.45 [tmsyringe filters (Whatman Puradisc 25 PP syringe filters with 0.45 [tmpolypropylene media) using a 60 mL disposable syringe (BD). The filteredemulsion was then vialed in Wheaton 5 mL serum sample vials with a 5 mLfill and stoppered and sealed with rubber stoppers and Flip-Off seals(West Pharmaceutical). Post formulation HPLC analysis showed thepaclitaxel concentration at 1.85 mg/mL.

The study design is shown in the following table, and the tumor size andbody weights are shown in table 2. See also, Curves 310, 320, 330, and340, FIG. 3.

TABLE 2 Treatment Regimen Group n Agent mg/kg Route Schedule 1 10 NoTreatment — — — 2 10 Non-targeted PTX 40 iv qd × 5 3 10 aVb3-targetedPTX 40 iv qd × 5 4 10 paclitaxel 15 iv qd × 5

There was no excessive toxicity in this study. No group had a meanweight loss greater than 20%. The mean tumor responses and body weightsare shown in FIG. 5. There is a statistically significant differencebetween the non-targeted and the targeted arms, with the targeted armshowing efficacy that is statistically insignificantly different thanthe positive control. The mean tumor volume at the study endpoint forthe untargeted nanoparticle formulation was 726 mm³ and the mean tumorvolume for the targeted nanoparticle formulation was 446 mm³. Thesestatistically significant differences demonstrate the added value oftargeting the nanoparticle emulsion.

Example 5 Demonstration of a Cisplatin-Containing Emulsion

In order to demonstrate the ability to incorporate a water-soluble smallmolecule therapeutic into the nanoparticle emulsion, a formulationcontaining cisplatin was achieved as follows:

1.03 g sodium phosphate monobasic monohydrate acid (Mallinckrodt) wasdissolved in approximately 950 mL sterile water for injection (OmniPur)in a 1 L beaker using magnetic stirring. Next 22.00 g glycerin(Mallinckrodt) was added with stirring. The pH of the buffer solutionwas then adjusted to final pH of 6.50 using 1 N sodium hydroxide (VWR).Finally the solution was brought to a final volume of 1 L using sterilewater for injection (OmniPur).

Next, 50.0 mg of cisplatin (CDDP; Tecoland) was suspended in 7 mL ofsafflower oil (Spectrum) by vortexing a 4 dram vial containing thecisplatin and safflower oil for 30 seconds then immersing it in a heated(41° C.) sonicating bath for 15 min. The vial and its contents were thenvortexed again for 30 seconds immediately prior to use.

0.70 g of purified egg phosphatidylcholine (Lipoid), 150 mg ofdipalmitoyl-sn-glycero-3- phosphoglycerol (DPPG; Lipoid) and 150 mg of1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550] (DSPE-PEG550; Avanti) were homogenized in 41 mL of pH 6.507.5 mM phosphate buffer with 22 mg/mL glycerin for 30 seconds in a 100mL disposable beaker using an Ultra-Turrax T25 rotor stator homogenizer.Next, 7 mL of safflower oil with 50.0 mg of cisplatin suspended in itwas added to the contents of the beaker, followed by the addition of 2mL of triacetin (Spectrum) and the combined components homogenized foran additional 2 minutes 30 seconds. The resulting crude emulsion wasthen processed on a M-110-Y Microfluidizer (Microfluidics) atapproximately 16,000 psi for 5 minutes with ice water being used to coolthe heat exchanger of the Microfluidizer. The fine emulsion was thensterile filtered through a single 32 mm 0.20 [tm syringe filter (PallAcrodisc PF syringe filter with 0.2 [tm Supor polyethersulfone media)using a 60 mL disposable syringe (BD). The sterile-filtered emulsion wasthen vialed in Wheaton 5 mL serum sample vials with a 5 mL fill andstoppered and sealed with rubber stoppers and Flip-Off seals (WestPharmaceutical). Post formulation HPLC analysis showed the cisplatinconcentration at 0.47 mg/mL.

Negative control vehicle was formulated as follows: 1.03 g sodiumphosphate monobasic monohydrate acid (Mallinckrodt) was dissolved inapproximately 950 mL sterile water for injection (OmniPur) in a 1 Lbeaker using magnetic stirring. Next 22.00 g glycerin (Mallinckrodt) wasadded with stirring. The pH of the buffer solution was then adjusted tofinal pH of 6.50 using 1 N sodium hydroxide (VWR). Finally the solutionwas brought to a final volume of 1 L using sterile water for injection(OmniPur).

0.70 g of purified egg phosphatidylcholine (Lipoid), 150 mg ofdipalmitoyl-sn-glycero3- phosphoglycerol (DPPG; Lipoid) and 150 mg of1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550] (DSPE-PEG550; Avanti) were homogenized in 41 mL of pH 6.507.5 mM phosphate buffer with 22 mg/mL glycerin for 30 seconds in a 100mL disposable beaker using an Ultra-Turrax T25 rotor stator homogenizer.Next 7 mL of safflower oil (Spectrum) was added to the contents of thebeaker, followed by the addition of 2 mL of triacetin (Spectrum) and thecombined components homogenized for an additional 2 minutes 30 seconds.The resulting crude emulsion was then processed on a M-110-YMicrofluidizer (Microfluidics) at approximately 16,000 psi for 5 minuteswith ice water being used to cool the heat exchanger of theMicrofluidizer. The fine emulsion was then sterile filtered through asingle 32 mm 0.20 [tm syringe filter (Pall Acrodisc PF syringe filterwith 0.2 [tm Supor polyethersulfone media) using a 60 mL disposablesyringe (BD). The sterile-filtered emulsion was then vialed in Wheaton 5mL serum sample vials with a 5 mL fill and stoppered and sealed withrubber stoppers and Flip-Off seals (West Pharmaceutical).

Next, demonstration of efficacy of this formulation was carried out onOVCAR3 human ovarian carcinoma xenograft models using female CB.17 SCIDmice, the results of which are shown in Curves 410, 420, and 430, FIG.4. In this three-arm study, a vehicle control was included, whichconsisted of the nanoparticle emulsion with no cisplatin. A positivecontrol arm included a conventional formulation of free cisplatin dosedat 4 mg/kg. The test arm included the nanoparticle emulsion at a dose of4 mg/kg.

60 female CB.17 SCID mice, aged 8 to 12 weeks, were implanted with—1 mm³OVCAR3 tumor fragments in the flank. When the tumors had grown toapproximately 100-150 mm³, pairs were matched by tumor size andtreatment began. Dosing was carried out weekly on all three arms, for atotal of four doses. Body weight and tumor size for each of the mice wastaken every other day. Mean tumor size and normalized mean body weightsare shown in FIG. 5. No excess toxicity was noted, as evidenced by bodyweight and observed behavior of the mice. The cisplatin nanoparticleformulation (labeled HJ9 in FIG. 6) effected a significant reduction intumor growth, as did the positive control of free cisplatin. Thenegative control (labeled ‘vehicle’ in FIG. 6) served as a reference fortumor growth in the absence of cisplatin.

Example 6 Treatment of Cancer in Humans

In accordance with the invention described herein, a prophetic exampleof its utility in the treatment of cancer in humans is provided.

A nanoparticle emulsion is formulated substantially as described inexample 1. Initial human safety trials are conducted with thenanoparticle emulsion according to standard clinical protocols todetermine maximum tolerated dose (MTD) in humans. Efficacy studies arethen conducted in humans to determine optimal dosing schedules forvarious types of cancer, where optimal dosing is determined by acombination of toxicity, tolerability, and tumor response. Dosingschedules may include intensive daily dosing, metronomic dosing (lowerdoses administered daily over extended periods of time), or lessfrequent but higher doses, as determined by clinical responses observedin the efficacy studies.

Patients with cancer types and sub-types that respond to the describednanoparticle formulation are then given appropriate treatment andmonitored for response. Patients may receive multiple treatmentsconsistent with acceptable toxicity and continued tumor response.

Example 7 Alternative Formulations

In a first alternative example, formulation of a nanoparticle emulsionwas formulated as described in example 1, except the ratio of saffloweroil to triacetin was 1:1 instead of 7:2 (safflower oil in excess). Thecrude emulsion was formulated using the microfluidization processdescribed above, and the fine emulsion was characterized with respect toparticle size. Immediately after formulation it was found that the meanparticle diameter was over 200 nanometers, with a very asymmetricdistribution that included a significant population of particles greaterthan 1 micrometer in diameter. Pools of free lipids were observedshortly after formulation, and within a day most of the nanoparticleshad disintegrated, as demonstrated by static light scatteringmeasurements, and there was visible phase separation in the bulkformulation.

In a second alternative example, formulation of a nanoparticle emulsionwas attempted as described in example 1, except that pure linoleic acidwas used instead of safflower oil. Linoleic acid is the predominantlipid in safflower oil, so it was anticipated that it might be a usefulsubstitute for safflower oil. The initial steps were carried out asdescribed in example 1, but during formulation of the crude emulsionusing high shear mixing there was significant foaming that preventedfurther processing to produce the fine emulsion. The formulation processwas aborted at this point. Subsequent attempts to produce a nanoparticleemulsion with pure linoleic acid have proven unsuccessful.

In a third alternative example, formulation of a nanoparticle emulsionwas attempted as described in example 1, except that pure oleic acid wassubstituted for the safflower oil. The initial steps were carried out asdescribed in example 1, but during formulation of the crude emulsionusing high shear mixing there was significant foaming that preventedfurther processing to produce the fine emulsion. The formulation processwas aborted at this point. Subsequent attempts to produce a nanoparticleemulsion with pure oleic acid have proven unsuccessful.

In a fourth alternative example, formulation of a nanoparticle emulsionwas attempted as described in example 1, except that no safflower oilwas used, just triacetin as the lipid component. The initial steps werecarried out as described in example 1, but it was not possible toproduce even a crude emulsion as the triacetin came out of solution tooquickly and formed distinct phase separation. Subsequent attempts toproduce a nanoparticle emulsion with pure triacetin acid have provenunsuccessful.

In a fifth alternative example, formulation of a nanoparticle emulsionwas attempted as described in example 1, except that no triacetin wasused, just safflower oil as the lipid component. The initial steps werecarried out as described in example 1, but it was not possible dissolveenough paclitaxel in the safflower oil to be clinically useful. Themaximum solubility was approximately 1000× less than in triacetin.Although it was possible to produce a stable fine nanoparticle emulsionwith just safflower oil as the lipid component, suggesting possibleformulations for APIs that are soluble in pure safflower oil, the amountof paclitaxel incorporated into the fine emulsion was far below what isrequired for therapeutic efficacy.

In a sixth alternative example, formulation of a nanoparticle emulsionwas attempted as described in example 1, except that the surfactantcommixture consisted entirely of DPPC and the ratio of safflower oil totriacetin was 8:3 instead of 7:2. A crude emulsion was successfullyformulated as described in example 1. However, the microfluidizationmethod of formulating the fine emulsion resulted in a preponderance ofparticles that were larger than 1 micrometer, and which were unstable.

In a seventh alternative example, formulation of a nanoparticle emulsionwas attempted as described in example 1, except that the surfactantcommixture consisted entirely of DPPC and the ratio of safflower oil totriacetin was kept at 7:2. A crude emulsion was successfully formulatedas described in example 1. However, the microfluidization method offormulating the fine emulsion resulted in a preponderance of particlesthat were larger than 200 nanometers, and which was unable to besterile-filtered.

While the preferred embodiments of the present invention have beenillustrated in detail, it should be apparent that modifications andadaptations to those embodiments may occur to one skilled in the artwithout departing from the scope of Applicants' disclosure.

We claim:
 1. A method to form a nanoparticle pharmaceutical deliverysystem, the method comprising: dissolving at least one activepharmaceutical ingredients (APIs) in at least one lipid to form alipid/API mixture; mixing the lipid/API mixture with an aqueous bufferand at least one first surfactant to form a crude emulsion, wherein theat least one first surfactant isolates the lipid/API mixture in theaqueous buffer; and processing the crude emulsion using high shearforces until a fine emulsion in the form of nanoparticles suspended inthe aqueous buffer is achieved.
 2. The method of claim 1 furthercomprising adding a targeting ligand either to the fine emulsion or tothe crude emulsion, wherein the targeting ligand binds to an outermostportion of the nanoparticles suspended in the aqueous buffer.
 3. Themethod of claim 2, wherein the targeting ligand is selected from thegroup consisting of an antibody, a peptidomimetic, a molecule havingfewer than 10 carbon atoms, and a therapeutic antibody.
 4. The method ofclaim 1, wherein the step of processing the crude emulsion furthercomprises maintaining a temperature of the crude emulsion at about 5° C.5. The method of claim 1, wherein the at least one API comprises ataxane or a platinum derivative.
 6. The method of claim 5, wherein theat least one API comprises paclitaxel or cisplatin.
 7. The method ofclaim 1, wherein the at least one API has a concentration in thetargeted nanoparticle pharmaceutical delivery system of about 0.1% toabout 0.6% (w/v).
 8. The method of claim 1, wherein the at least onelipid comprises triacetin.
 9. The method of claim 1, wherein the atleast one lipid comprises safflower oil.
 10. The method of claim 1,wherein the at least one lipid has a concentration in the targetednanoparticle pharmaceutical delivery system of about 12% to about 28%(v/v).
 11. The method of claim 1, wherein the at least one surfactanthas a concentration in the targeted nanoparticle pharmaceutical deliverysystem of about 1.5% to about 5.0% (w/v).
 12. The method of claim 1,wherein the at least one lipid comprises triacetin and safflower oil,wherein a volume ratio between the triacetin and the safflower oil isbetween about 13:4 and about 7:1.
 13. The method of claim 1, wherein theat least one surfactant is comprised in a surfactant commixture, thesurfactant commixture further comprising at least one second surfactantand a targeting ligand.
 14. A nanoparticle delivery system, formed bythe following process: dissolving at least one active pharmaceuticalingredient (API) in at least one lipid to form a lipid/API mixture;mixing the lipid/API mixture with at least one first surfactant and anaqueous buffer to form a crude emulsion, wherein the at least one firstsurfactant isolates the lipid API mixture in the aqueous buffer; andprocessing the crude emulsion using high shear forces until a fineemulsion in the form of nanoparticles suspended in the aqueous buffer isachieved.
 15. The nanoparticle delivery system of claim 14 furthercomprising adding a targeting ligand to the fine emulsion, wherein thetargeting ligand binds to an outermost portion of the nanoparticlessuspended in the aqueous buffer.
 16. The nanoparticle pharmaceuticaldelivery system of claim 14, wherein the step of processing the crudeemulsion further comprises maintaining a temperature of the crudeemulsion at about 5° C.
 17. The nanoparticle pharmaceutical deliverysystem of claim 14, wherein the at least one API comprises a taxane or aplatinum derivative.
 18. The nanoparticle pharmaceutical delivery systemof claim 17, wherein the at least one API comprises paclitaxel orcisplatin.
 19. The nanoparticle pharmaceutical delivery system of claim14, wherein the at least one surfactant is comprised in a surfactantcommixture, the surfactant commixture further comprising at least onesecond surfactant and a targeting ligand.
 20. The nanoparticlepharmaceutical delivery system of claim 14, wherein the at least onelipid comprises triacetin.
 21. The nanoparticle pharmaceutical deliverysystem of claim 14, wherein the at least one lipid comprises saffloweroil.
 22. The nanoparticle pharmaceutical delivery system of claim 14,wherein the at least one API has a concentration in the nanoparticlepharmaceutical delivery system of about 0.1% to about 0.6% (w/v). 23.The nanoparticle pharmaceutical delivery system of claim 14, wherein theat least one lipid comprises triacetin and safflower oil, wherein avolume ratio between the safflower oil and the triacetin is betweenabout 13:4 and about 7:1.
 24. The nanoparticle pharmaceutical deliverysystem of claim 14, wherein the at least one lipid has a concentrationin the nanoparticle pharmaceutical delivery system of about 12% to about28% (v/v).
 25. The nanoparticle pharmaceutical delivery system of claim14, wherein the at least one surfactant has a concentration in thenanoparticle pharmaceutical delivery system of about 1.5% to about 5.0%(w/v).